A functional nervous system relies on the formation of precise synaptic connections between specific target neurons. Two key elements of excitatory synapse formation are the formation of mature, mushroom-shaped dendritic spines and the recruitment of synaptic proteins, like NMDARs and PSD-95. A number of adhesion molecules, including EphB/ephrinB, synCAM, SALM2, NGL2, neurexin/neuroligin and cadherins, have each been shown to regulate aspects of synaptogenesis. However, the molecular mechanisms that underlie synapse formation as well as how factors act to coordinate synapse formation in the developing animal remain to be determined. To begin to address these issues, we have focused on the role of the EphB family of receptor tyrosine kinases and their ephrinB ligands because EphB knockout mice show serious deficits in synapse formation, while available knockouts for other synaptic adhesion molecules appear not to have an excitatory synapse formation phenotype. Moreover, because cocaine alters expression of EphB and ephrinB in cortex and striatum, our work on mechanisms of synaptogenesis will provide insights into cortical development and impact our understanding of disease and addiction. We propose that trans-synaptic EphB- ephrinB signaling induces both pre- and post-synaptic organization of multiple components of synapses: presynaptic ephrinB, through interactions with dendritic EphB, stimulates postsynaptic development while the reciprocal interaction of postsynaptic EphB induces axonal ephrinB dependent clustering of presynaptic proteins. I propose three specific aims to address the role of EphB and ephrinB in synapse development. Specific aim 1: Determine the signaling mechanisms responsible for EphB-dependent induction of presynaptic specializations. Specific aim 2: Test whether EphB acts to initiate synapse formation. Specific aim 3: Determine the role of EphB proteins in specifying dendritic locations of cortical synaptic contacts in vivo. Our approach of moving from more reduced model systems to the intact animal is innovative and will lead to significant new findings. Moreover, given the role of EphB/ephrinB in synaptic and structural plasticity, and the sensitivity of EphBs and ephrinBs to drugs of abuse, our studies will provide fundamental insights into mechanisms controlling synapse development and with the advent of cell based therapies will advance understanding of human developmental diseases and addiction.